The present invention relates to methods and compositions for the direct detection of analytes using color changes that occur in immobilized biopolymeric material in response to selective binding of analytes to their surface.
A major goal of analyte detection research is to develop inexpensive, fast, reliable, and sensitive detectors. Unfortunately, the technologies developed to date have only met some of these goals, and no single device has sufficiently attained a majority of them.
Classical detection methods such as liquid chromatography (LC), gas chromatography (GC), and supercritical fluid chromatography (SFC), in combination with mass spectrometry, are widely used and provide accurate identification of analytes and quantitative data. However, these techniques are time consuming, extremely expensive, require sample preconcentration, and are difficult or impossible to adapt to field use.
Biosensors (i.e., devices containing biological material linked to a transducing apparatus) have been developed to overcome some of the shortcomings of the classical analyte detection techniques. Many currently used biosensors are associated with transducer devices that use photometry, fluorimetry, and chemiluminescence; fiber optics and direct optical sensing (e.g., grating coupler); surface plasmon resonance; potendiometric and amperometric electrodes; field effect transistors; piezoelectric sensing; and surface acoustic wave (Krxc3xa4mer, J. AOAC Intern. 79: 1245 [1996]). However, there are major drawbacks to these devices, including their dependence on a transducing device, which prevents miniaturization and requires a power source. These disadvantages make such devices too complex, expensive, or unmanageable for many routine analyte detection applications such as field work or home use. Additionally, many of these devices are limited by the lack of stability and availability of the biological materials (e.g., proteins, antibodies, cells, and organelles).
Immunoassay methods are also used for detecting certain types of analytes. In these methods, antibodies are developed to specifically bind to a target of interest (e.g., an analyte). By labeling the antibody (e.g., with dye or fluorescent or radioactive material), binding of the antibody to an analyte can be detected. However, immunoassay methods are limited in that they require production of antibodies against each analyte of interest. Antibodies cannot be generated against some types of analytes and their generation can be time consuming and expensive.
The art remains in need of analyte detectors that provide the specificity of biosensors but also provide the cost-efficiency, stability, accuracy, reliability, reproducibility, and robustness that is lacking from available technologies. In particular, development of devices that can be miniaturized with controlled shapes and that do not rely on an energy source would also be very beneficial, particularly for routine field work and home use.
The present invention relates to methods and compositions for the direct detection of analytes using color changes that occur in immobilized biopolymeric material in response to selective binding of analytes to their surface.
The present invention provides various methods and compositions useful for the detection of analytes.
In one embodiment, the present invention provides methods for immobilizing biopolymeric material: providing a metal oxide, biopolymeric material, an acid, a buffer, and a sonicating means; sonicating the metal oxide and the acid using the sonicating means to produce a sonicated solution; adding the buffer to the sonicated solution to produce a buffered solution; and adding the biopolymeric material to the buffered solution to produce an organic/inorganic solution.
In alternative embodiments of the methods, the present invention further comprises the steps of applying the organic/inorganic solution to a formation support to produce a formed organic/inorganic solution; and gelling the formed organic/inorganic solution to produce an organic/inorganic device.
In preferred embodiments, the metal oxide comprises tetramethylorthosilicate, although it is contemplated that any material that can be used to produce substantially transparent, porous glass will be used in the methods of the present invention.
In some embodiments, the biopolymeric material is selected from the group consisting of liposomes, films, multilayers, braided, lamellar, helical, tubular, and fiber-like shapes, solvated rods, solvated coils, and combinations thereof.
In other embodiments, the biopolymeric material comprises a plurality of self-assembling monomers selected from the group consisting of diacetylenes, acetylenes, alkenes, thiophenes, polythiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes anilines, pyrroles, vinylpyridinium, and combinations thereof, although any self-assembling monomer that will form biopolymeric material is contemplated by the present invention. In preferred embodiments, the diacetylenes are selected from a group consisting of 5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid, 5,7-pentacosadiynoic acid, and combinations thereof. In yet other embodiments, the self-assembling monomers contain head groups selected from the group consisting of carboxylic acid, hydroxyl groups, amine groups, amino acid derivatives, and hydrophobic groups, although any head group that exists or can be synthesized on self-assembling monomers is contemplated by the presently claimed invention.
In some preferred embodiments, the biopolymeric material further comprises a ligand. In some embodiments, the ligand is selected from the group consisting of peptides, carbohydrates, nucleic acids, biotin, drugs, chromophores, antigens, chelating compounds, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, antibodies, and combinations thereof, although any ligand that can be linked to, or associated with, biopolymeric material is contemplated by the present invention.
In some preferred embodiments, the acid comprises hydrochloric acid, while in other preferred embodiments, the buffer comprises 3-[N-Morpholino]propanesulfonic acid. In other embodiments, the sonicating is conducted at a temperature from 0xc2x0 C. to 20xc2x0 C.
The present invention further provides an organic/inoiganic device produced according to any and all of the methods described above. In addition, the present invention provides biopolymeric material encapsulated in sol-gel glass. In some embodiments, the glass comprises tetramethylorthosilicate.
In some preferred embodiments, the biopolymeric material encapsulated in sol-gel glass is selected from the group consisting of liposomes, films, multilayers, braided, lamellar, helical, tubular, and fiber-like shapes, solvated rods, solvated coils, and combinations thereof. In particularly preferred embodiments, the biopolymeric material comprises self-assembling monomer selected from the group consisting of diacetylenes, acetylenes, alkenes, thiophenes, polythiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes anilines, pyrroles, vinylpyridinium, and combinations thereof, although any self-assembling monomer that will form biopolyrneric material is contemplated by the present invention. In preferred embodiments, the diacetylenes are selected from a group consisting of 5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid, 5,7-pentacosadiynoic acid, and combinations thereof. In yet other embodiments, the self-assembling monomers contain head groups selected from the group consisting of carboxylic acid, hydroxyl groups, amine groups, amino acid derivatives, and hydrophobic groups, although any head group that exists or can be synthesized on self-assembling monomers is contemplated by the presently claimed invention.
In some preferred embodiments, the biopolymeric material encapsulated in sol-gel glass further comprises a ligand. In some embodiments, the ligand is selected from the group consisting of peptides, carbohydrates, nucleic acids, biotin, drugs, chromophores, antigens, chelating compounds, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, antibodies, and combinations thereof, although any ligand that can be linked to, or associated with, biopolymeric material is contemplated by the present invention.
The present invention further provides methods for detecting analytes: providing biopolymeric material encapsulated in sol-gel glass, a detection means, and one or more analyte; exposing the biopolymeric material encapsulated in sol-gel glass to the analyte to produce a response; and detecting said response using the detection means.
In preferred embodiments, the glass comprises tetramethylorthosilicate, although it is contemplated that any material that can be used to produce substantially transparent, porous glass will be used in the methods of the present invention.
In some embodiments, the biopolymeric material is selected from the group consisting of liposomes, films, multilayers, braided, lamellar, helical, tubular, and fiber-like shapes, solvated rods, solvated coils, and combinations thereof.
In other embodiments, the biopolymeric material comprises a plurality of self-assembling monomers selected from the group consisting of diacetylenes, acetylenes, alkenes, thiophenes, polythiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes anilines, pyrroles, vinylpyridinium, and combinations thereof, although any self-assembling monomer that will form biopolymeric material is contemplated by the present invention. In preferred embodiments, the diacetylenes are selected from a group consisting of 5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid, 5,7-pentacosadiynoic acid, and combinations thereof. In yet other embodiments, the self-assembling monomers contain head groups selected from the group consisting of carboxylic acid, hydroxyl groups, amine groups, amino acid derivatives, and hydrophobic groups, although any head group that exists or can be synthesized on self-assembling monomers is contemplated by the presently claimed invention.
In some preferred embodiments, the biopolymeric material further comprises a ligand. In some embodiments, the ligand is selected from the group consisting of peptides, carbohydrates, nucleic acids, biotin, drugs, chromophores, antigens, chelating compounds, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, antibodies, and combinations thereof, although any ligand that can be linked to, or associated with, biopolymeric material is contemplated by the present invention.
In some embodiments, the analyte is selected from the group consisting of small molecules, pathogens, bacteria, membrane receptors, membrane fragments, enzymes, drugs, antibodies, and combinations thereof, although any analyte that can be detected through its interaction with a ligand or the biopolymeric material is contemplated by the present invention.
In yet other embodiments, the biopolymeric material encapsulated in sol-gel glass comprises a badge.
In preferred embodiments, the detection means is selected from the group consisting of visual inspection, spectrometry, optical fiber, quartz oscillators, electrode surfaces, and scintillation, although any detection means that provides analysis of the presence of an analyte is contemplated by the present invention.
In some embodiments, the response is used as a competitive binding measurement to quantitate and characterize the presence of natural binding sites. In other embodiments, the biopolymeric material encapsulated in sol-gel glass comprises an array.